Characteristic Face: A Key Indicator for Direct Diagnosis of 22q11.2 Deletions in Chinese Velocardiofacial Syndrome Patients Dandan Wu1., Yang Chen1., Chen Xu1, Ke Wang2, Huijun Wang3, Fengyun Zheng4, Duan Ma3,4*, Guomin Wang1* 1 Department of Oral & Cranio-maxillofacial Science, Shanghai 9th People’s Hospital, College of Stomatology, School of Medicine, Shanghai Jiao Tong University, Shanghai Key Laboratory of Stomatology, Shanghai, P. R. China, 2 Department of Oral and Maxillofacial Surgery, The Affiliated Hospital, Medical School, Qingdao University, Qingdao, P. R. China, 3 Children’s Hospital, Fudan University, Shanghai, P. R. China, 4 Key Laboratory of Molecular Medicine, Ministry of Education, Department of Biochemistry and Molecular Biology, Institute of Medical Sciences, Shanghai Medical College, Fudan University, Shanghai, P. R. China Abstract Velocardiofacial syndrome (VCFS) is a disease in human with an expansive phenotypic spectrum and diverse genetic mechanisms mainly associated with copy number variations (CNVs) on 22q11.2 or other chromosomes. However, the correlations between CNVs and phenotypes remain ambiguous. This study aims to analyze the types and sizes of CNVs in VCFS patients, to define whether correlations exist between CNVs and clinical manifestations in Chinese VCFS patients. In total, 55 clinically suspected Chinese VCFS patients and 100 normal controls were detected by multiplex ligation-dependent probe amplification (MLPA). The data from MLPA and all the detailed clinical features of the objects were documented and analyzed. A total of 44 patients (80.0%) were diagnosed with CNVs on 22q11.2. Among them, 43 (78.2%) presented with 22q11.2 heterozygous deletions, of whom 40 (93.0%) had typical 3-Mb deletion, and 3 (7.0%) exhibited proximal 1.5-Mb deletion; no patient was found with atypical deletion on 22q11.2. One patient (1.8%) presented with a 3-Mb duplication mapping to the typical 3-Mb region on 22q11.2, while none of the chromosomal abnormalities in the MLPA kit were found in the other 11 patients and 100 normal controls. All the 43 patients with 22q11.2 deletions displayed characteristic face and palatal anomalies; 37 of them (86.0%) had cognitive or behavioral disorders, and 23 (53.5%) suffered from immune deficiencies; 10 patients (23.3%) manifested congenital heart diseases. Interestingly, all patients with the characteristic face had 22q11.2 heterozygous deletions, but no difference in phenotypic spectrum was observed between 3-Mb and 1.5-Mb deletions. Our data suggest that the characteristic face can be used as a key indicator for direct diagnosis of 22q11.2 deletions in Chinese VCFS patients. Citation: Wu D, Chen Y, Xu C, Wang K, Wang H, et al. (2013) Characteristic Face: A Key Indicator for Direct Diagnosis of 22q11.2 Deletions in Chinese Velocardiofacial Syndrome Patients. PLoS ONE 8(1): e54404. doi:10.1371/journal.pone.0054404 Editor: Bart Dermaut, Pasteur Institute of Lille, France Received August 19, 2012; Accepted December 11, 2012; Published January 16, 2013 Copyright: ß 2013 Wu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by the funds from National Natural Science Foundation of China (No. 81070813), Nation Science Supporting Plan (No. 2006BAI5A09), and Hospital Foundation of Shanghai Ninth People’s Hospital (No. 201212). The funders had no role in study design, data collectionand analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] (GW); [email protected] (DM) . These authors contributed equally to this work. Introduction the disease from parents, while most of them are sporadic (de novo) due to a heterozygous deletion caused by nonallelic homologous VCFS, also known as DiGeorge syndrome, conotruncal recombination (NAHR), mainly mediated by the low copy repeats anomalies face syndrome, and CATCH 22, is firstly termed by (LCRs) on 22q11.2. Three types of deletion on 22q11.2 have been Shprintzen et al. in 1978 and exhibits an expansive phenotype with reported so far. While most the cases have typical 3-Mb deletion more than 180 clinical features involving almost every organ and with breakpoints flanked by LCR A and D, some cases carry system [1,2]. The major symptoms include congenital heart proximal 1.5-Mb deletion flanked by LCR A and B within the disease, particular conotruncal malformation, characteristic face, typically deleted region (TDR); only a few cases show atypical palatal abnormality, immune deficiency, and cognitive or behav- deletions overlapping or nonoverlapping with the TDR [5]. ioral disorder. Its minor features involve growth retardation, Researchers also found that certain cases with 22q11.2 duplication neonatal hypocalcemia, feeding difficulty, hearing loss, limb or CNVs on other chromosomes, such as 4q, 8p, 10p, and 17p, deformity, and so on. The penetrance of each clinical feature is presented with clinical phenotypes similar to those of VCFS; these different; no single phenotype occurs in all patients, and none is cases were also defined as VCFS by some geneticists and obligatory [3]. clinicians, who believed that such cases increased the diversity of VCFS is also one of the most common human genomic genetic mechanism of VCFS [6–10]. disorders with a population prevalence ranging from approxi- mately 1:2000 to 1:7000 [4]. Only about 5–15% patients inherit PLOS ONE | www.plosone.org 1 January 2013 | Volume 8 | Issue 1 | e54404 Molecular Diagnosis of Chinese VCFS Patients Although recent studies have improved our understanding of analyzed in GeneMarker software V1.8 (Softgenetics, State the pathogenesis of VCFS, the correlations between the pheno- College, PA). types and genotypes of CNVs remain ambiguous [11]. While The MLPA kit used in this study contained 48 probes for 48 discordant types of CNVs can occur in the cases with identical different genes. There were 29 probes targeting 22q11 region, phenotypes, concordant chromosomal aberrations may result in among which 9 probes were between LCR A and B; 3 probes variable expressions, even within families or between homozygotic between LCR B and C; 2 probes between LCR C and D; 3 probes twins [12,13]. Thus, exactly determining the specified clinical between LCR D and E; 4 probes between LCR E and F; 2 probes features for each type of CNVs will facilitate genetic counseling between LCR F and G; 1 probe between LCR C and D; and 5 and health care. probes in Cat Eye syndrome region proximal to LCR A. The This study has detected 55 clinically-suspected Chinese VCFS other 19 probes located in other locus outside 22q11. Among patients by MLPA, with 100 healthy subjects as control, aiming to them, 2 probes were on 4q34-qter; 3 probes on 8p23; 2 probes on reveal the types and sizes of CNVs in VCFS patients with different 9q34.3; 6 probes on 10p14; 4 probes on17p13.3; and 2 probes on phenotypes and explore the correlations between CNVs and 22q13. clinical findings in Chinese VCFS patients. Each sample was detected in triplicate by MLPA, and the replicates were detected on different days. Methods Results Patients All 55 patients enrolled in this study were from the Center for Of the 55 patients, 44 (80.0%) were confirmed with CNVs on Cleft Lip and Palate, Shanghai Ninth People’s Hospital, Shanghai 22q11.2 by MLPA analysis. Among them, 43 cases (78.2%) Jiao Tong University School of Medicine, and all the clinical showed 22q11.2 heterozygous deletion, of whom 40 (93.0%) findings in these patients were confirmed with physical or auxiliary exhibited typical 3-Mb deletion with breakpoints between LCR A examinations by specialists. Patients from No. 1–43 commonly and D, while 3 (7.0%) displayed proximal 1.5-Mb deletion presented with characteristic face and palatal abnormality, and between LCR A and B; no case was found with atypical deletion most of them displayed other features such as congenital heart on 22q11.2. Additionally, 1 case (1.8%) had 3-Mb duplication diseases, immune deficiency, and cognitive or behavioral disor- mapping to the typical 3-Mb region on 22q11.2 (Figure 2 and 3). ders. Patients No. 44 presented with palatal abnormality, immune None of the chromosomal abnormalities in the MLPA kit were deficiency, mild mental retardation, low set ears, and conductive detected in the rest 11 patients and 100 normal controls. hearing disturbance. Patients No. 45–54 commonly presented According to the clinical features, all 55 VCFS patients (100%) with congenital heart diseases and palatal abnormality, and some had palatal anomalies; 43 patients (78.2%) exhibited characteristic of them showed other features too. Patients No. 55 presented with face; 40 patients (72.7%) displayed cognitive or behavioral palatal abnormality, mild mental retardation, and conductive disorders; 24 patients (43.6%) had immune deficiency; and 20 hearing disturbance (Table 1). The identified congenital heart patients (36.4%) suffered from congenital heart diseases. Other diseases included tetralogy of Fallot, interrupted aortic arch, features with lower frequencies were ear deformities, conductive ventricular septal defect, atrial septal defect, and patent ductus hearing disturbance, exotropia, cleft lip, tooth hypocalcification, arteriosus. The characteristic face consisted of vertically long